Immunoassay is an analytical technique widely used in medicine and the biological sciences. The term "immunoassay" as used herein encompasses analytical methods for detecting, locating or quantifying biological substances by use of a label. Generally a label, such as radioactive isotope, is attached to a molecule of the substance of interest. The presence of the labeled molecule can then be detected by suitable means.
There are various types of immunoassay in common use. In one type of immunoassay, a sample, e.g., a solution, containing both an unknown and a labeled antigen of interest is incubated with an antibody specific for that antigen. If the unknown also contains the antigen, then both the labeled and unlabeled antigens compete for binding sites on the antibody. The antibody can be immobilized on a solid support, such as a test tube, glass beads, latex particles, etc. Incubation is followed by a separation step in which the antigen bound to the antibody on the support is separated from unbound antigen. Through measurement of the amount of bound labeled antigen, the presence and/or quantity of similar, unlabeled antigen can be determined. That is, the higher the concentration of antigen in the unknown, the fewer the binding sites occupied by a labeled antigen. Thus, the detected level of the labeled antigen (e.g., counts per minute of radioactivity) is an inverse function of the concentration of the unlabeled antigen.
A second type of immunoassay is known as sandwich immunoassay. In this method, an antibody rather than an antigen is labeled. A sample containing an unknown is incubated with an immobilized antibody. Antigens, if present in the sample, will bind to the antibody. After incubation, unbound material is removed by a separation step. In a second incubation with a solution of labeled antibody, the bound antigen is "sandwiched" between the immobilized antibody and the labeled antibody which adheres to the antigen. After a second separation, the amount of labeled antibody is determined. Detection of labeled antibody is indicative of the presence of antigen.
In general, the most commonly used type of label is a radioactive substance, such as .sup.125 I, which can easily and accurately be detected. However, materials labeled radioactively often have a short shelf life, both because of radioactive decay of the label and because radiation degrades the labeled molecule. Further, handling of radioactive substances entails risks to laboratory personnel.
The present invention is directed to novel compounds which are useful as labels in luminometric immunoassay (hereinafter referred to as "LIA") in both conventional and sandwich assay techniques. Unlike radioimmunoassay, LIA does not employ radioactive materials. Rather, the label attached to the antigen or antibody is a chemiluminescent compound, that is, a compound which can undergo a reaction (usually oxidative) in which light is a product. The light emission is measured by appropriate devices, and in certain cases, the light intensity is indicative of the quantity of labeled material.
Known chemiluminescent substances suitable for use in immunoassays include luminol (5-amino-2, 3-dihydrophthalazine-1, 4-dione), isoluminol (6-amino-2, 3-dihydrophthalazine-1, 4-dione) and the various acridinium esters.